The overall goal of our research is to uncover the molecular genetic alterations involved in the development of hereditary and sporadic human cancer. Our main objectives are to characterize the genetic changes in cancer cells, and to evaluate normal gene function and pathogenesis of disease. Ultimately, knowledge about the molecular basis of tumor development will facilitate the establishment of more effective ways to prevent, diagnose and treat cancer.
Recently, we have turned our attention to the study of hereditary colorectal cancer (CRC). CRC is the second leading cause of death from cancer in Western countries. Every year, approximately 130,000 new cases are diagnosed and 50,000 CRC-related deaths occur in the United States. A conspicuous fraction of these cases (5 to 15%) is the consequence of a genetic predisposition. The two main familial CRC syndromes are hereditary nonpolyposis CRC (HNPCC, or Lynch Syndrome) and familial adenomatous polyposis (FAP).
HNPCC is an autosomal dominant disorder characterized by early onset colorectal tumors that are frequently multiple, and may be associated with extracolonic malignancies such as cancers of the endometrium, stomach, ovary, brain, skin and urinary tract. Patients affected by HNPCC carry a germline mutation in one of the genes involved in long-patch DNA mismatch repair (MMR), such as MSH [mutS homolog] 2, MLH [mutL homolog] 1, MSH3, GTBP [G/T mismatch-binding protein]/MSH6, PMS [postmeiotic segregation] 2 and PMS1. MMR is a specialized system that corrects base-base mismatches, short insertions/deletions and recombination-derived heteroduplexes, thus contributing to mutation avoidance and genetic stability.
In an effort to identify novel genes involved in DNA repair or related DNA transactions, we employed the yeast interaction trap. One of the MLH1 interactors that we identified is a novel human DNA repair gene, which we named MED1. The MED1 gene encodes a protein with tripartite structure, containing an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific base excision repair (BER) glycosylases/lyases. For its structure, MED1 represents the first indication that methylation of the genome on cytosines at CpG sites has a novel and hitherto poorly characterized role in mammalian cells (besides transcriptional repression, imprinting, X-chromosome inactivation, recombination and suppression of parasitic sequences), i.e., participation in DNA repair.
We and others have previously shown that MED1 (also named MBD4) acts as a G:T and G:U mismatch-specific thymine and uracil glycosylase. MED1 activity prefers G:T and G:U mismatches located in the context of methylated and unmethylated CpG sites. G:T and G:U mismatches originate via spontaneous hydrolytic deamination of 5-methylcytosine and cytosine to thymine and uracil, respectively. MED1 is likely involved in the repair of deaminated 5-methylcytosine and cytosine at CpG sites. The absence of MED1 function would lead, in the next round of DNA replication, to G:C>A:T transition mutations; these changes at CpG sites are the most frequent mutations in human cancer.
Patients affected by FAP develop hundreds or thousands of colorectal polyps (or adenomas), which may evolve into malignant cancer. In addition, these patients are predisposed to the development of gastric and duodenal polyps, desmoid tumors and osteomas and, in some cases, hepatoblastomas. FAP patients carry a germline mutation in one allele of the tumor suppressor gene APC, which acts as a "gatekeeper" of normal proliferation in the colon. Somatic loss of the second APC allele in colonic cells leads to adenoma formation. However, in order to identify the earliest molecular changes in colorectal tumorigenesis, it is necessary to analyze colonic cells bearing a single-hit in the APC tumor suppressor gene (heterozygous state). For this reason, we have recently initiated microarray-based studies to characterize the mRNA expression profile of the morphologically normal mucosa of FAP patients, in comparison to the normal mucosa of control individuals.